y-IFN (CB[3-gal). This allowed the transduction efficiency to be determined by enumeration of 
blue cells, after staining with X-gal (65) The data in Fig 14 indicate that three different human 
melanoma cells are all readily transduced with amphotropic retroviral vector. 
3. 3. 3. 2. y-IFN AND HLA EXPRESSION IN HUy VECTOR-TRANSDUCED HUMAN 
MELANOMA CELLS. Huy retroviral vector was used to introduce the human y-IFN gene into a 
number of short term melanoma tumor cell lines. Cell-free supernatants from G418 selected 
transduced human melanoma cell lines were tested for y-IFN activity. The data in Table VI 
indicate that biologically active y-IFN is expressed in transduced cells. 
To determine the effect of gene transfer of y-IFN on human Class I MHC (HLA) expression, cell 
extracts were prepared and analyzed for HLA expression by protein immunoblotting. The data in 
Fig 15 indicate that transduction of human y-IFN vector into three different human melanoma cell 
lines resulted in a 2-7-fold increase in the level of MHC (HLA I), as determined by protein 
immunoblotting. Immunofluorescence indicates that this is surface associated (data not shown). 
These stably transduced G418 resistant tumor cells were then examined by flow cytometry for 
changes in the expression of HLA Class I and II encoded gene products as indicated by W6/32 and 
L243 staining, respectively. Representative data for two such lines (Table VII) demonstrate the 
substantial increases in the level of cell surface expression of Class I HLA proteins following 
transduction. Although the percent positivity of the two representative cell lines for Class I HLA is 
not measurably changed relative to the parental cells, the level of expression in gene modified DM6 
of these important proteins is increased 1.6-fold and by gene modified DM265 by more than 2.6- 
fold. With respect to Class II HLA proteins, gene modified DM6 tumor cells have a 5-fold 
increase in percent positivity. Gene transduced DM265 tumor cells demonstrate a small increase in 
frequency of positives but a measurable 1.4-fold increase in the level of expression of Class II 
proteins. The data indicate that transduction of these tumor cdl lines (and other such tumor cells 
not shown) results in substantial and long lasting upregulation of both Class I and Class II HLA 
proteins. 
3. 3. 3. 3. RECOGNITION OF GENE MODIFIED MELANOMA BY HLA RESTRICTED, 
MELANOMA SPECIFIC, CYTOTOXIC T-CELLS. We have examined the functional nature of the 
increased HLA proteins on the gene transduced tumor cell lines. DM6 and DM6/y-IFN tumor cells 
were tested in vitro as stimulator cells in a ^H-thymidine uptake assay using an HLA-A2 restricted, 
melanoma specific cytotoxic T-cell line as a responding population. While native DM6 tumor cells 
elicit a measurable response, the gene modified DM6/y-IFN are dramatically more effective (6- to 
40-fold) in triggering the T-cells (Table VIII). These data (Table VIII) clearly demonstrate the 
superiority of y-IFN enhanced DM6/y-IFN to elicit a T-cell proliferative response. 
Recombinant DNA Research, Volume 17 
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